Nitric Oxide Contributes to Behavioral, Cellular, and Developmental Responses to Low Oxygen in Drosophila  James A. Wingrove, Patrick H. O'Farrell  Cell  Volume 98, Issue 1, Pages 105-114 (July 1999) DOI: 10.1016/S0092-8674(00)80610-8 Copyright © 1999 Cell Press Terms and Conditions

Figure 1 Hypoxia Induces Exploratory Behavior in Larvae (A–E) Exposure of early third instar larvae to hypoxia (1% O2) resulted in a rapid behavioral response in which the larvae ceased feeding and extracted themselves from the food. Images were taken at 10 s intervals following onset of hypoxia (A–E). The panels show a mound of yeast in which the larvae are largely buried. Initially, only their posterior ends protrude (arrows in [B] and [C]), but they turn around as they prepare to leave the food (arrows in [D] and [E] show larvae with their more pointed anterior ends protruding). Scale bar is 5 mm. (F) After 15 min of hypoxia, approximately 75% of the larva had left the food. Percents given are the average of three experiments. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions

Figure 2 NO and PKG Are Involved in Hypoxia-Induced Motility (A and B) Larvae carrying different for alleles have altered PKG levels (see text) and behavioral responses to hypoxia. (A) shows an experiment in which equal number of fors and forR larvae were subjected to 1% oxygen for 10 min. The larvae were previously fed colored food so that forR larvae are green (green arrows) and fors larvae are red (red arrows). (B) summarizes the results of three experiments in which an average of 48% of forR larvae left the yeast after 10 min of hypoxia (48.7% ± 7.5%) compared to 13% of fors larvae (13.2% ± 6.7%). (C and D) Larvae fed NOS inhibitor are deficient in hypoxia-induced motility. (C) is an example of an experiment in which an equal number of larvae fed the NOS inhibitor L-NAME (red larvae, red arrows) or the inactive isomer D-NAME (green larvae, green arrows) were subjected to 10 min of 1% oxygen. (D) summarizes the results of three trials in which 52% (52.3% ± 14.1%) of the D-NAME-fed larvae left the yeast after 10 min, compared to 18% of the L-NAME-fed larvae (18.2% ± 8.5%). (E and F) Induced expression of a NOS transgene results in hypersensitivity to hypoxia. (E) is an example of an experiment in which an equal number of either wild-type larvae (red larvae, red arrows) or larvae containing a hs-iNOS transgene (green larvae, green arrows) were subjected to heat shock for 20 min at 37°C, allowed to recover for 60 min, and then subjected to 10% O2 for 15 min. (F) shows the average of three trials in which wild-type and nonheat-shocked iNOS stocks showed low percentages of motility at 10% O2 (wt = 20.3% ± 3.2%; hs − wt = 12.3% ± 3.5%; iNOS = 16.3% ± 3.2%), whereas heat-shocked iNOS showed an increased sensitivity to hypoxia with nearly 60% (59.3% ± 4%) of the larvae leaving the yeast after 15 min of 10% O2. The scale bars in (A), (C), and (E) are 5 mm. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions

Figure 3 Localization of NO and NOS Activity in Larvae (A–C) NOS activity is present in the larval CNS and spiracles. Diaphorase staining (dark blue) was utilized to visualize the presence of NOS activity in various tissues following dissection of third instar larvae. (A) shows a disc (labeled i.d.) and the larval CNS (labeled CNS), both of which are lightly stained. (B) shows posterior spiracles that have been dissected with attached cuticle and trachea. Large cells at the base of the posterior spiracles stain intensely. (C) shows anterior spiracles that have been dissected from a larva with some surrounding cuticle. A structure immediately proximal to the end of the tracheal tubes is heavily stained. The inset at the top right in (B) shows the posterior portion of a larva with an arrow indicating the posterior spiracles; in (C), the anterior portion of a larva is shown with an arrow indicating the anterior spiracles. (D) shows an anterior spiracle stained with DAF2/DA; arrows indicate neuronal-like processes and cell bodies that also stain intensely with the dye. Scale bar is 50 μm. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions

Figure 4 The dg2 Gene Is Involved in Surviving Prolonged Hypoxia (A) The dg2 gene is required in embryos for survival during prolonged hypoxia. Stage 12 forR and fors embryos were subjected to hypoxia for either 2, 4, 6, or 12 hr and then removed and allowed to develop at 25°C on grape agar plates for 30 hr. The percent viability was determined by the number of larvae that hatched. Viability of forR embryos: 0 hr, 96%; 2 hr, 92% ± 2.8%; 4 hr, 90% ± 2.9%; 6 hr, 93% ± 5%; 12 hr, 97% ± 2.9%. Viability of fors embryos: 0 hr, 89%; 2 hr, 89% ± 8.7%; 4 hr, 82% ± 8.6%; 12 hr, 18% ± 5.8%. Percentages are the average of three experiments. (B) The dg2 gene is required in larvae for survival during prolonged hypoxia. Early third instar forR and fors larvae were subjected to hypoxia for 6 hr, after which they were removed and allowed to develop on grape agar plates containing yeast. The percent viability was determined by the number of flies that eclosed. Viability of forR larvae, 83% ± 11.1%; fors larvae, 24% ± 4.0%. Percentages are the average of three experiments. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions

Figure 5 NO and PKG Are Utilized in a Hypoxic Block to S Phase (A and B) Hypoxia blocks S phase. Wild-type Sevelen embryos (stage 8) show BrdU incorporation into S phase cells during a 5 min labeling period (A). This incorporation is blocked when embryos are labeled from 5 to 10 min following imposition of hypoxia. (C and D) The hypoxic block to S phase was mimicked by the ectopic expression of iNOS. Embryos (stage 10) carrying a hs-iNOS transgene were exposed to heat shock for 30 min at 37°C, allowed to recover, and then assayed for S phase using BrdU (D). Control embryos treated similarly (not shown) or nonheat-shocked transgenic embryos (C) had typical patterns of BrdU incorporation (Edgar and O'Farrell 1990). (E and F) The addition of the NO donor SNAP to stage 12 embryos resulted in a complete inhibition of BrdU incorporation. Embryos were exposed to 5 mM SNAP for 15 min prior to labeling with BrdU (F). Control embryos were left untreated (E). (G and H) The addition of 8Br-cGMP to stage 14 embryos also resulted in a reduction of S phase as assayed by BrdU incorporation. Embryos were incubated in 5 mM 8Br-cGMP for 60 min prior to treatment with BrdU to detect S phase (H). Control embryos were similarly treated but incubated with 5 mM GMP (G). (I–N) Inhibition of NO or PKG either genetically or pharmacologically resulted in a failure to block S phase during hypoxia. CNS (optic lobes shown) dissected from forR larvae showed a block to S phase when exposed to hypoxia (I and K), whereas CNS dissected from fors larvae continued to progress through S phase under hypoxia (J and L). Stage 11/12 embryos (360–480 min AED) that were incubated in 0.1 M D-NAME prior to hypoxia showed a block to S phase during hypoxia (M), whereas incubation of embryos in 0.1 M L-NAME allowed for continued BrdU incorporation during hypoxia (N). Scale bar is 50 μm. In (A–H) and (M and N), anterior is to the left and dorsal toward the top. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions

Figure 6 NO and PKG Promote Tracheal Branching in Larvae (A and B) fors larvae are deficient in tertiary tracheal branching. Anterior midguts immediately posterior to the proventriculus were dissected from early third instar forR (A) and fors (B) larvae and mounted in PBS with 5% Ficoll. Arrows indicate tertiary branches. Scale bar is 50 μm. (C and D) Ectopic expression of iNOS increases tertiary tracheal branching. Early second instar Sevelen (C) and hs-iNOS (D) larvae were subjected to 30 min of heat shock at 37°C and then allowed to age for 12 hr followed by another 30 min heat shock. Twelve hours later, third instar larvae midguts were dissected and mounted as described in (A) and (B). Arrows indicate tertiary branches. (E and F) Inhibition of NOS blocks tertiary tracheal branching. Wild-type larvae were fed from hatching either 0.5 M of D-NAME (E) or L-NAME (F). Midguts from early third instar larvae were dissected and mounted as described in (A and B). Arrows indicate tertiary branching. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions

Figure 7 NO and PKG Play Multiple Roles in the Response to Hypoxia in Drosophila A model suggesting how NO and PKG might be utilized by Drosophila to sense and respond to hypoxia. Unknown upstream factors might sense oxygen levels and affect levels of NO. Alternatively, the pathway might be activated at different levels in response to hypoxia. Our data suggest that the NO/PKG pathway influences multiple responses (depicted at the bottom of the figure) to hypoxia in Drosophila. The responses were altered by genetically or pharmacologically activating or inhibiting the pathway (manipulations are shown boxed in gray). Note that the pathway shown (dashed line) is inferred from the pathway established in other systems. Cell 1999 98, 105-114DOI: (10.1016/S0092-8674(00)80610-8) Copyright © 1999 Cell Press Terms and Conditions